Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: The level of the interaction between cBRC3 and cRAD51 was half of that observed with cBRC4 and similar to that with hBRC3 and hBRC4. The interaction between hRAD51 and cBRC3 was stronger than with hBRC3. (A) VP16 transactivation domain-fused cRAD51 construct or empty vector and a Gal4-DBD-fused cBRC3, cBRC4, hBRC3, hBRC4, or cBRCA2 N-terminus (1–1000 aa) were introduced into HeLa cells to determine their interaction with cRAD51 in a mammalian two-hybrid luciferase-based assay. HeLa cells were co-transfected with the BRC repeat constructs and cRAD51 expression vector constructs with the reporter plasmids pGluc and pRL-TK. The pRL-TK construct was used to normalize the transfection efficiency. Lysate luciferase activity was determined 48 h after transfection. (B) same as (A), except that VP16 transactivation domain-fused hRAD51 was used. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test, followed by a Tukey's post-test. Different letters indicate significant differences between the cells transfected with the indicated constructs. (p<0.01). DBD, DNA-binding domain; TAD, transactivation domain-fused proteins.

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Construct, Plasmid Preparation, Luciferase, Transfection, Expressing, Activity Assay, Binding Assay

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) Amino acid sequence alignment of BRC3 among canines (Accession No. NP_001006654.2), humans (Accession No. NP_000050.2), and mice (Accession No. NP_033895.2). (B) Amino acid sequence alignment of cBRC3 and hBRC4. The pink characters indicate the positions of missense mutations in cBRC3. (C–E) The contacts between the residues of 1526T (C), T1526P (D), or T1526A (E) and hRAD51 or other residues of BRC4 were calculated and are depicted. Human BRC4 and hRAD51 are depicted by gold and pink ribbons, respectively. Solid green lines signify stable contacts as determined by the University of California, San Francisco Chimera software.

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Sequencing, Software

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) The GST pull-down assay showed that wild-type and K1435R mutant canine BRC3 (cBRC3) interacted with RAD51, but the T1425P mutant cBRC3 did not interact with RAD51. GST-fused peptides were incubated with human RAD51 (hRAD51) at 4°C for 1 h. Equilibrated beads were incubated with the protein–peptide complexes at 4°C for 1 h. Complexes bound to the beads were thoroughly washed. The samples were analyzed by SDS-PAGE and western blotting with anti-RAD51 IgG, or anti-GST IgG, and horseradish peroxidase conjugated anti-rabbit IgG. Blots were developed using the ECL Plus Western Blotting Detection System. (B) Schematic of the enzyme-linked immunosorbent assay (ELISA) used to detect the cBRC-hRAD51 interaction and its disruption by soluble peptides. (C) In a competitive ELISA, GST-cBRC3 with the T1425P mutation did not inhibit the solid-phase interaction between GST-cBRC3 and hRAD51. However, the substitution of GST-cBRC3 with the K1435R mutation showed no interaction with hRAD51, like wild-type cBRC3. Values are expressed as the mean percentage absorbance at 405 nm for data sets in triplicate normalized to the positive control, which was not treated with the soluble inhibitors, and the negative control, which was coated with GST and treated with hRAD51. Significance was examined by a one-way analysis of variance test followed by Tukey's post-test. The asterisks indicate a significant difference compared with GST-treated data sets at the same competitor concentration (p<0.01).

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Pull Down Assay, Mutagenesis, Incubation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Positive Control, Negative Control, Concentration Assay

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: The binding intensity of T1425P mutant canine BRC3 (cBRC3) and canine RAD51 (cRAD51) or human RAD51 (hRAD51) was much lower than that of wild-type cBRC3 and cRAD51 or hRAD51, while that of the K1435R mutant cBRC3 and cRAD51 or hRAD51 was higher than between wild-type cBRC3 and cRAD51 or hRAD51. (A) Gal4-DNA binding domain-fused wild-type and mutant cBRC3 or mutants of human BRC4 (hBRC4) and VP16 transactivation domain-fused cRAD51 constructs were introduced into HeLa cells to determine their interaction with cRAD51 in a mammalian two-hybrid assay measuring luciferase activity. HeLa cells were co-transfected with the BRC repeat constructs and cRAD51 expression vector constructs and with the reporter plasmids pGluc and pRL-TK. The pRL-TK construct was used to normalize the transfection efficiency. Lysate luciferase activity was determined 48 h after transfection. (B) same as (A), except that VP16 transactivation domain-fused hRAD51 was used. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared with cells transfected with wild-type (WT) cBRC3 (p<0.01).

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Binding Assay, Mutagenesis, Construct, Two Hybrid Assay, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) Schematic of the modified mammalian two-hybrid assay used to examine how mutant canine BRC3 (cBRC3) interferes with the RAD51-RAD51 interaction. (B) The graph shows the strength of the interference caused by the expression of EGFP empty vector, wild-type EGFP-cBRC3, 2 mutants of cBRC3 (T1425P and K1435R), and cBRCA2 N-terminus (1–1000 aa) on the canine RAD51 (cRAD51)-cRAD51 interaction as determined by the modified mammalian two-hybrid assay. Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared with the cells transfected with an empty vector (*: p<0.05, **: p<0.01). The negative control for this assay is shown in Supporting information . (C) same as (B), except that human RAD51 (hRAD51) was used. (D) Immunostained cells, which were transiently transfected with cBRC3 mutants, after being subjected to ionizing radiation, are shown. The indicated cells were irradiated (15 Gy) and fixed 12 h after exposure to the ionizing radiation. (E) HeLa cells transfected with wild-type or mutant cBRC3 or empty vector were irradiated (15 Gy) and then allowed to recover for the times indicated. Images containing at least 100 cells were captured by a computer, and the number of cells containing at least 10 foci were recorded and plotted as a percentage of the total number of cells. The plots were generated from 3 independent experiments. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared with irradiated cells transfected with an empty vector at the same time point (p<0.01).

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Modification, Two Hybrid Assay, Mutagenesis, Expressing, Plasmid Preparation, Transfection, Negative Control, Irradiation, Generated

Journal: PLoS ONE

Article Title: Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

doi: 10.1371/journal.pone.0045833

Figure Lengend Snippet: (A) Amino acid sequence alignment among canine BRC3 (cBRC3) (Accession No. NP_001006654.2), wild-type human BRC3 (hBRC3) (Accession No. NP_000050.2), and 2 mutants of hBRC3. The pink characters indicate the positions of missense mutations in cBRC3. (B) Gal4-DNA binding domain-fused wild-type and mutant hBRC3 and VP16 transactivation domain-fused cRAD51 constructs were introduced into HeLa cells to determine their interaction with cRAD51 in a mammalian two-hybrid assay measuring luciferase activity. HeLa cells were co-transfected with the hBRC3 constructs and canine RAD51 (cRAD51) expression vector constructs and with the reporter plasmids pGluc and pRL-TK. The pRL-TK construct was used to normalize the transfection efficiency. Lysate luciferase activity was determined 48 h after transfection. The results are given as the mean (± standard error) (n = 3). Significance was examined by a one-way analysis of variance test followed by Dunnett's post-test. The asterisks indicate a significant difference compared to cells transfected with wild-type (WT) cBRC3 (*: p<0.05, **: p<0.01). (C) same as (B), except that VP16 transactivation domain-fused hRAD51 was used. (D) The graph shows the strength of the interference caused by the expression of EGFP empty vector, wild-type EGFP-hBRC3, 3 mutants of hBRC3 (K1440R, K1440E, and T1430P), and cBRCA2 N-terminus (1–1000 aa) on the cRAD51-cRAD51 interaction as determined by the modified mammalian two-hybrid assay. Significance was examined by one-way ANOVA test followed by Dunnett's test. The asterisks indicate a significant difference compared to the cells transfected with an empty vector (*: p<0.05, **: p<0.01). The negative control for this assay is shown in Supporting information . (E) same as (D), except that hRAD51 was used.

Article Snippet: HeLa cell monolayers (80% confluent) cultured on coverslips (Matsunami Glass, Osaka, Japan) were transfected with the NLS-pEGFP-C1 vectors containing wild-type or mutant cBRC3 by using FuGENE HD (Promega, Madison, WI, USA).

Techniques: Sequencing, Binding Assay, Mutagenesis, Construct, Two Hybrid Assay, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation, Modification, Negative Control

Journal: Scientific Reports

Article Title: MUC4-ErbB2 Oncogenic Complex: Binding studies using Microscale Thermophoresis

doi: 10.1038/s41598-019-53099-0

Figure Lengend Snippet: Schematic view of MUC4. Schematic representation of the membrane-bound mucin MUC4 featuring both subunits MUC4α and MUC4β. MUC4α is the mucin-like subunit, with a 27 amino-acid (aa) long signal peptide, an approximate 126–130 aa repeat domain, a 554 aa domain and a 16 aa sequence, variably repeated from 145 to 345 times and rich in proline/serine/threonine residues (PST domain), strongly O-glycosylated. Two protein domains are then found, the AMOP and the NIDO domains. The subunits are post-transcriptionally cleaved by an auto-cleavable GDPH sequence and non-covalently associated. MUC4β is formed by a von Willebrand factor-D (VWD) followed by an uncharacterized part, strongly N-glycosylated and by a cysteine-rich domain (CR). Two EGF-like domains come next, EGF3 and EGF1 separated by a short linker. Then an intermediate uncharacterized domain (DI), another EGF-like domain EGF2 and a transmembrane helix (TM) with a short cytoplasmic tail (CT).

Article Snippet: MUC4 EGF3+1+2 construct was designed and produced by ProteoGenix SAS with the same specifications, considering the domain limits from Uniprot (from the start of EGF1 domain to the end of EGF2 domain), based on data from our previous work .

Techniques: Sequencing

Journal: Scientific Reports

Article Title: MUC4-ErbB2 Oncogenic Complex: Binding studies using Microscale Thermophoresis

doi: 10.1038/s41598-019-53099-0

Figure Lengend Snippet: Effect of different combinations of lysis/dilution buffer on the binding curve of MUC4-ErbB2 in MST. Combinations of lysis/dilution buffer are given in this order. Binding curves of eGFP-MUC4β with ErbB2-Fc at 23 °C in RIPA-MST (pink curve), in RIPA-PBS (green curve) or MPer-MST (dark red curve) lead to K d > 20 µM with no saturation. The binding curve in MPer-PBS condition (orange) allowed to find a K d value in the range of several hundred nanomolars.

Article Snippet: MUC4 EGF3+1+2 construct was designed and produced by ProteoGenix SAS with the same specifications, considering the domain limits from Uniprot (from the start of EGF1 domain to the end of EGF2 domain), based on data from our previous work .

Techniques: Lysis, Binding Assay

Journal: Scientific Reports

Article Title: MUC4-ErbB2 Oncogenic Complex: Binding studies using Microscale Thermophoresis

doi: 10.1038/s41598-019-53099-0

Figure Lengend Snippet: Improved binding curves for interaction of eGFP-MUC4β and ErbB2-Fc in normal and reverse mode. ( A ) Dose-response curve in triplicate for the binding interaction between ErbB2-Fc and eGFP-MUC4β lysate, with a dilution ratio of 3:1 instead of 1:1, all other parameters being kept optimal. The curve obtained presents two distinguishable binding events that can be approximated with a Hill model fitting. The first (green curve) is around 25 ± 5 nM and the second (red curve) spawn at 152 ± 80 nM. ( B ) Dose-response curve in triplicate for the binding interaction between ErbB2-Fc tagged by red fluorescent anti-HIS dye and eGFP-MUC4β lysate, with a dilution ration of 3:1. MUC4 concentrations range from 2.27 nM to 403 nM. The curve obtained presents two distinguishable binding events that can be approximated with a Hill model fitting. The first (green curve) is around 7 ± 5 nM and the second (red curve) spawns at 55 ± 17 nM.

Article Snippet: MUC4 EGF3+1+2 construct was designed and produced by ProteoGenix SAS with the same specifications, considering the domain limits from Uniprot (from the start of EGF1 domain to the end of EGF2 domain), based on data from our previous work .

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: MUC4-ErbB2 Oncogenic Complex: Binding studies using Microscale Thermophoresis

doi: 10.1038/s41598-019-53099-0

Figure Lengend Snippet: Dose-response binding curves in normal and reverse mode for interaction of eGFP-MUC4 EGF3+1+2 and ErbB2-Fc. The blue curve corresponds to the normal mode, following the fluorescence of the eGFP tagged protein with titration of ErbB2-Fc. The green curve corresponds to the reverse mode with ErbB2 tagged to be red fluorescent and titrated against eGFP-MUC4 EGF3+1+2 . While the concentration of eGFP-MUC4 EGF3+1+2 protein is kept constant at 33 nM, the ErbB2-Fc concentration ranged from 1 μM and 0.05 nM. While the concentration of ErbB2-Fc is kept constant at 50 nM, the eGFP-MUC4 EGF3+1+2 concentration ranged from 4,9 nM to 370 nM. The normal mode binding curve yields a K d of 79 ± 18 nM while the reverse mode yields a K d of 65 ± 14 nM.

Article Snippet: MUC4 EGF3+1+2 construct was designed and produced by ProteoGenix SAS with the same specifications, considering the domain limits from Uniprot (from the start of EGF1 domain to the end of EGF2 domain), based on data from our previous work .

Techniques: Binding Assay, Fluorescence, Titration, Concentration Assay

Journal: Cell Death & Disease

Article Title: Sequential Cdk1 and Plk1 phosphorylation of protein tyrosine phosphatase 1B promotes mitotic cell death

doi: 10.1038/cddis.2012.208

Figure Lengend Snippet: PTP1B undergoes phosphorylation during mitotic arrest induced by Taxol (TAX) and Nocodazole (NOC). K562 and KYO1 cells were untreated (UNT), treated with DMSO (0.1% v/v; VEH) and ( a ) increasing concentrations of NOC and TAX (0.01–1 μ M) for 12 h or ( b ) NOC (1 μ M) and TAX (1 μ M) for the indicated time points. Whole-cell extracts (WCEs) were prepared and equal amounts of protein were resolved by SDS-PAGE and probed with anti-PTP1B antibody and anti-GAPDH antibody was used as a loading control. ( c ) K562 cells were treated with DMSO (0.1% v/v), NOC (1 μ M) or TAX (1 μ M) for 12, 24, 36 and 48 h. DNA content was analysed by flow cytometry following propidium iodide (1 μ g/ml) staining and the percentage of cells containing 4n and <2n DNA was calculated. ( d ) K562 cells were either left UNT or treated with DMSO (0.1% v/v), NOC (1 μ M) or TAX (1 μ M) for 12 h. Ultracentrifugation was used to separate mitochondrial, microsomal, and cytosolic fractions, and a WCE was retained as a control. Equal amounts of protein were resolved by SDS-PAGE, and probed with anti-PTP1B antibody by western blotting. Anti-calnexin, anti-GAPDH and anti-MnSOD antibodies were used as a microsomal, cytosolic and mitochondrial markers, respectively. ( e ) K562 cells were transfected with pEGFP-c1-PTP1B (1 μ g) and incubated at 37 °C for 24 h prior to staining with DAPI (1 μ g/ml) and MitoTracker (100 nM), and images were acquired by fluorescent microscopy. ( f ) K562 cells were treated either DMSO (0.1% v/v) or TAX (1 μ M) for 12 h. Mitochondrial protein (50 μ g) was incubated with CIP for 30 min at 37 °C, in the absence or presence of the Na 3 VO 4 . Protein was resolved by SDS-PAGE and probed with anti-PTP1B antibody. Anti-MnSOD antibody was used as a mitochondrial loading control

Article Snippet: K562 cells were transfected with PTP1B–pEGFP-c1 vector and mutants, using the Nucleofector II 96-well shuttle system (Lonza Group Ltd., Basel, Switzerland), as directed by the manufacturer.

Techniques: SDS Page, Flow Cytometry, Staining, Western Blot, Transfection, Incubation, Microscopy

Journal: Cell Death & Disease

Article Title: Sequential Cdk1 and Plk1 phosphorylation of protein tyrosine phosphatase 1B promotes mitotic cell death

doi: 10.1038/cddis.2012.208

Figure Lengend Snippet: Cdk1 co-immunoprecipitates with PTP1B and phosphorylates it at serine 386. ( a ) K562 cells were synchronised at the G1/S border using a double thymidine block. At 1 h post-release, cells were treated with DMSO (0.1% v/v) or Taxol (TAX) (1 μ M) in the absence or presence of BI2536 (BI) (20 μ M), CK2 inhibitor (CK) (20 μ M) or RO-3306 (RO) (9 μ M) for a 2 h period beginning at 8, 9 or 10 h post-thymidine release. Protein was resolved by SDS-PAGE and probed with PTP1B, Bim, BubR1, and GAPDH antibodies. ( b ) PTP1B was immunoprecipitated from either untreated interphase (I) or mitotically-synchronised (M) K562 cells. Protein was resolved by SDS-PAGE and probed with anti-Cdk1 antibody. A portion (10%) was retained and probed with PTP1B to confirm immunoprecipitation. Blue arrow indicates PTP1B protein, red arrow indicates antibody heavy chain. ( c ) Purified recombinant WT-PTP1B-His (500 ng) was incubated with increasing amounts of recombinant Cdk1/cyclin B1 (0–460 ng), 2 μ Ci of [ γ - 32 P] ATP, ATP (100 μ M), at 30 °C for 1 h. The reaction was terminated by the addition of Laemmli buffer to 1 × concentration. ( d ) Purified recombinant PTP1B-His (WT, S386A, S386E, and L317G) (500 ng), was incubated with recombinant Cdk1/cyclin B1 (200 ng), ATP (100 μ M) and 2 μ Ci of [ γ - 32 P] ATP for 1 h at 30 °C. The reaction was terminated by the addition of Laemmli buffer to 1 × concentration. Protein was resolved by SDS-PAGE, and [ γ - 32 P] incorporation was measured by autoradiography after gel drying. The amount of Plk1 and PTP1B was visualised by CBB staining. ( e ) Amount of [ γ - 32 P] incorporation in ( d ) was normalised to WT PTP1B levels and expressed as normalised intensity of [ γ - 32 P] incorporation. Results are representative of the mean±S.E.M. of three independent experiments ( *= P <0.05, **= P <0.01, using a paired T -test)

Article Snippet: K562 cells were transfected with PTP1B–pEGFP-c1 vector and mutants, using the Nucleofector II 96-well shuttle system (Lonza Group Ltd., Basel, Switzerland), as directed by the manufacturer.

Techniques: Blocking Assay, SDS Page, Immunoprecipitation, Purification, Recombinant, Incubation, Concentration Assay, Autoradiography, Staining

Journal: Cell Death & Disease

Article Title: Sequential Cdk1 and Plk1 phosphorylation of protein tyrosine phosphatase 1B promotes mitotic cell death

doi: 10.1038/cddis.2012.208

Figure Lengend Snippet: Plk1 phosphorylates PTP1B following a priming phosphorylation by Cdk1. ( a ) PTP1B and ( b ) Plk1 were immunoprecipitated from mitotically-synchronised K562 cells. Immunoprecipitated protein was resolved by SDS-PAGE and probed with Plk1 or PTP1B, respectively. In each case a portion of the sample was retained to confirm immunoprecipitation. Blue arrow indicates PTP1B, red arrow indicates antibody heavy chain. ( c ) Purified recombinant WT-PTP1B-His (500 ng) was incubated with increasing amounts of recombinant Plk1 (0–460 ng), 2 μ Ci of [ γ - 32 P] ATP, and ATP (100 μ M), at 30 °C for 1 h. The reaction was terminated by the addition of Laemmli buffer to 1 × concentration. As a control, Plk1 (460 ng) was incubated with either WT-PTP1B-His (500 ng) or Cdc25C (500 ng) and 2 μ Ci of [ γ - 32 P] ATP, and ATP (100 μ M), at 30 °C for 1 h, as before. ( d ) Purified WT-PTP1B-His (500 ng) was incubated in a one-step kinase assay either alone (NEG) or in the presence of Cdk1 (460 ng) or Plk1 (460 ng) for 1 h at 30 °C (lanes 1–3) or pre-incubated with Cdk1 (460 ng) or Plk1 (460 ng) prior to incubation with the alternate kinase, 2 μ Ci of [ γ - 32 P] ATP and ATP (100 μ M) in a two-step kinase assay as outlined (lanes 4–5). The reaction was terminated by the addition of Laemmli buffer. Protein was resolved by SDS-PAGE, and [ γ - 32 P] incorporation was measured by autoradiography after gel drying. The amount of Plk1 and PTP1B was visualised by CBB staining. Results are representative of three independent experiments

Article Snippet: K562 cells were transfected with PTP1B–pEGFP-c1 vector and mutants, using the Nucleofector II 96-well shuttle system (Lonza Group Ltd., Basel, Switzerland), as directed by the manufacturer.

Techniques: Immunoprecipitation, SDS Page, Purification, Recombinant, Incubation, Concentration Assay, Kinase Assay, Autoradiography, Staining

Journal: Cell Death & Disease

Article Title: Sequential Cdk1 and Plk1 phosphorylation of protein tyrosine phosphatase 1B promotes mitotic cell death

doi: 10.1038/cddis.2012.208

Figure Lengend Snippet: Cdk1-mediated phosphorylation of serine 386 is an essential priming step for Plk1-mediated phosphorylation at serine 286 and 393. ( a ) Purified recombinant PTP1B-His (WT, S386A, S386E) (500 ng), or Cdc25C (500 ng), was incubated with recombinant Cdk1/cyclin B1 (200 ng), ATP (100 μ M) and 2 μ Ci of [ γ - 32 P] ATP for 1 h at 30 °C. The reaction was terminated by the addition of Laemmli buffer to 1 × concentration. ( b ) Purified PTP1B-His (WT, or S386A, S286A, S393A, and S286A/S393A mutants) (500 ng) was incubated with ATP (100 μ M), in the presence of Cdk1 (460 ng) at 30 °C for 1 h. The reaction was terminated by incubation at 65 °C for 15 min. The reaction samples were chilled on ice for 5 min, and then incubated with Plk1 (460 ng), 2 μ Ci of [ γ - 32 P] ATP and ATP (100 μ M), for a subsequent kinase reaction at 30 °C for 1 h. The reaction was terminated by the addition of Laemmli buffer. Protein was resolved by SDS-PAGE, and [ γ - 32 P] incorporation was measured by autoradiography after gel drying. The amount of PTP1B was visualised by CBB staining. ( c ) The amount of [ γ - 32 P] incorporation was normalised to WT PTP1B levels, and expressed as normalised intensity of [ γ - 32 P] incorporation. Results are representative of the mean±S.E.M. of three independent experiments ( n =3, *= P <0.05, **= P <0.01, using a paired T -test)

Article Snippet: K562 cells were transfected with PTP1B–pEGFP-c1 vector and mutants, using the Nucleofector II 96-well shuttle system (Lonza Group Ltd., Basel, Switzerland), as directed by the manufacturer.

Techniques: Purification, Recombinant, Incubation, Concentration Assay, SDS Page, Autoradiography, Staining

Journal: Cell Death & Disease

Article Title: Sequential Cdk1 and Plk1 phosphorylation of protein tyrosine phosphatase 1B promotes mitotic cell death

doi: 10.1038/cddis.2012.208

Figure Lengend Snippet: PTP1B phosphorylation regulates mitotic cell death and PTP1B phosphatase activity. K562 cells (1 × 10 6 ) were transfected with pEGFP-c1 empty vector (1 μ g), WT PTP1B-EGFP-c1 (1 μ g), or S286A, S393A, and S286A/S393A mutants (1 μ g). Twenty-four hours post-transfection, cells were harvested and WCEs were prepared in RIPA lysis buffer. Equal amounts of protein (50 μ g) were resolved by SDS-PAGE and probed with anti-PTP1B antibody. Anti-GAPDH was used as a loading control ( a ). Transfected cells were treated with DMSO (VEH; 0.1% v/v), Nocodazole (NOC; 1 μ M) or Taxol (TAX; (1 μ M) for 24 h ( b-d ). DNA content was analysed by flow cytometry following propidium iodide staining. Results represent the mean±S.E.M. of three independent experiments (* P ≤0.05). ( e ) Purified recombinant PTP1B (WT, S286E, S393E, and S286E/S393E) (500 ng), were diluted 1 : 1 in phosphatase reaction buffer and incubated in duplicate with a well-coated poly-(Glu, pTyr) substrate for 1 h at 37 °C. Dephosphorylation of substrate was measured by incubation with an anti-pTyr antibody conjugated to HRP enzyme for 30 min at 37 °C, followed by reaction with the colorimetric HRP substrate TMBZ. The HRP reaction was stopped after 15 min incubation at 37 °C, by the addition of 6N H 2 SO 4 , and absorbance at 450 nm was measured. Absorbance values were normalised to units of phosphatase activity by comparison against the standard curve, and results were expressed as units/μg of PTP1B. Results represent the mean±S.E.M. of three independent experiments (* P ≤0.05)

Article Snippet: K562 cells were transfected with PTP1B–pEGFP-c1 vector and mutants, using the Nucleofector II 96-well shuttle system (Lonza Group Ltd., Basel, Switzerland), as directed by the manufacturer.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Lysis, SDS Page, Flow Cytometry, Staining, Purification, Recombinant, Incubation, De-Phosphorylation Assay

Journal: PLoS ONE

Article Title: Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell–Giant Unilamellar Vesicle Electrofusion

doi: 10.1371/journal.pone.0106853

Figure Lengend Snippet: (A) Plasmid DNA encoding EGFP (pEGFP; CMV promoter, 4.7 kbp) was adopted as a reporter. pEGFP was introduced into HeLa cells and cells were then cultured for 1 or 5 days. From the top row, images obtained by fluorescent microscopy are shown for GUV-treated cells after culturing for 1 day, 5 days, cells lacking GUVs (1 day), or not exposed to the DC pulse (1 day). Phase contrast (left column), EGFP expression in cells (green, middle column), and merged images (phase contrast shown in red-scale; right column) are shown. Scale bar = 50 µm. (B) An artificially designed DNA nanostructure (origami) was also introduced into HeLa cells. The DNA origami structure was labeled with green fluorescent dye (by using FITC-conjugated oligonucleotides, 282 FITC molecules per single origami). Corresponding images of phase contrast (left column), DNA origami (green, middle column), and merged images (phase contrast shown in red-scale) were obtained by fluorescent microscopy immediately after cell–GUV electrofusion. Scale bar = 10 µm.

Article Snippet: An EGFP and mCherry expression vector (pEGFP-C1, pmCherry) were prepared using a NucleoBond Xtra Midi plus kit (Macherey-Nagel GmbH & Co., Düren, Germany), according to the manufacturer's instructions.

Techniques: Plasmid Preparation, Cell Culture, Microscopy, Expressing, Labeling, Electrofusion